Project Summary Middle ear infection or otitis media (OM) is the most predominant bacterial disease of childhood and results in significant morbidity in the U.S. and globally. Episodes of OM can substantially affect a child?s ability to hear normally. This loss of hearing is of great concern due to the long-term consequences on language development. Multiple different bacterial species and viruses cause middle ear infections. Nontypeable Haemophilus influenza (NTHI) is the primary bacterial pathogen of chronic OM, recurrent OM and OM wherein there is treatment failure. Several human pathogens, including NTHI, have evolved a novel genetic system, termed the phasevarion (for phase variable regulon), which mediates a rapid and reversible change in the expression of many genes throughout the chromosome. This epigenetic regulation occurs via phase variation of a single gene (modA) that encodes a DNA methyltransferase, and results in two phenotypically distinct subpopulations, ON and OFF. Our preliminary data clearly demonstrated that differences in the gene expression between these subpopulations have a significant affect on in vitro phenotypes known to be critical to the disease course of OM. We further showed that the status of the phasevarion, as well as switching from one status to the other, significantly affects disease severity in a chinchilla model of experimental OM. However, ModA2-regualted gene expression during ascension from the site of colonization, the nasopharynx, to the site of disease, the middle ear is unknown. In Specific Aim 1, we will assess site-specific expression of genes regulated by the ModA2 phasevarion in a model of ascending experimental model that mimics the disease course in children. Studies to determine gene expression within the host during experimental OM are absolutely necessary to fully understand the mechanisms by which the phasevarion contributes to both disease severity and bacterial persistence in various anatomical niches. Additionally, phasevarion regulation occurs through epigenetic methylation and cannot be discerned in silico. In Specific Aim 2, we will define immune cell recruitment, define cytokine expression profiles and identify antibodies developed against each modA2 population as well as a shift in ModA2 state over the course of disease development. A comprehensive understanding of when, where and how the phasevarion regulates expression of virulence factors combined with subsequent host immune responses to these changes in NTHI gene expression is required for the development of optimal disease treatment and prevention strategies. We expect to contribute significantly to this improved understanding of the role of the phasevarion in bacterial-host interactions via successful completion of Specific Aims of this proposal.